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PHOTOGRAPHIC QUANTIFICATION OF FLUORESCENTLY STAINED DNA IN GELS THROUGH ANALOG MODIFICATION OF SPECTROPHOTOMETER OUTPUT
PHOTOGRAPHIC QUANTIFICATION OF FLUORESCENTLY DNA IN GELS THROUGH ANALOG MODIFICATION OF SPECTROPHOTOMETER OUTPUT
STAINED
Charles E. Willis*, Jaime A. Oro+ and Horace B. Gray, Jr.* *Department of Biochemical and Biophysical Sciences and +Department of Electrical Engineering University of Houston, Houston, Texas 77004
INTRODUCTION Fluorescence photographs constitute a permanent record of the results of electrophoretic separations of nucleic acid molecules. Images in these photographs are produced by enhanced ethldium bromide fluorescence when this dye is bound to DNA or RNA and exposed to u.v, light. Though fluorograms are used routinely to locate DNA In g e l s , nonlinearity in the pho tographic film darkening function complicates determination of the actual distribution of fluorescent material from photographic images. However, fluorescence photography is an inexpensive alternative to both direct fluorometry and radioactive labeling, and offers greater sensitivity for nucleic acid detection than direct absorbance measurements. Moreover, the principles of fluorographic analysis are applicable to autoradiography. The simplest methods for evaluating fluorographic images assume a linear relationship between the mass of fluorescing compound and the peak height of an absorbance scan of the photographic negative (DeLeys and Jackson, 1967). A similar method involves integration of the absorbance peaks (Depew and Wang, 1975; Dugaiczyk and others, 1 9 7 5 ) . Both approaches are valid as long as the maximum absorbance observed is much less than the contrast of the film (Pulleyblank and others, 1 9 7 5 ) . This is because there is a region of the film darkening curve, at relatively low incident intensities, in which absorbance of the developed film is linear with inci dent intensity. The range of useful measurement can be extended by deter mining the film darkening response as a function of incident intensity (Prunell and others, 1977; Pulleyblank and others, 1 9 7 7 ) . The absorbance traces are transformed into fluorescence intensity profiles (hence, mass distributions) by means of this film darkening curve in which the absor bance varies linearly with the log of exposure. Though the transformation can be performed by digital computers, one novel method (Hörz and others, 1981) describes the use of analog modification of densitometer output assuming the above linear-log film darkening function. Though inexpensive to implement, the analog method of Hörz and others (1981) uses the absorbance signal from the densitometer in the exponent of Its transfer function and as such is extremely sensitive to the determined
381
382
BIOMEDICAL ENGINEERING II
value of background absorbance. Expressing the transfer function in terms of transmittance makes use of the proportionality I « [ΐ/Τ)1/ϊ
(1)
where I^ is the intensity of film exposure, Ί_ represents fraction of inci dent light transmitted by the developed film and γ is the contrast of the film (slope of the linear region of the absorbance vs_ log(exposure) curve). This relationship is just as simple to implement and also has minimum and maximum calibration points for the transmittance signal. All conventional methods for determining the film darkening function rely on external fluorescent standards. Since an equimolar series of molecular weight standards is generally included in gel electrophoresis experiments with DNA, internal standardization is possible. In order to use these standards, assumptions must be made about the distribution of DNA within the band corresponding to a particular fragment. Assuming a Gaussian distribution of DNA within each band and effective diffusion coefficients which depend on the one-halfth power of molecular weight, the peak absor bance within each of these bands is linearly related to the product of the contrast of the film darkening curve and the log of the molecular weight of the DNA. The less that "diffusion" depends on molecular weight, the closer that the slope of a plot of peak absorbance versus log(molecular weight) approximates contrast. When the Gaussian distribution function and the linear-log darkening func tion are used to predict the absorbance as a function of position in the band, the absorbance profile describes a parabola. This relationship should be valid except at the "tails" where the linear-log function fails to describe the film darkening response. Pulleyblank and others (1977) have suggested an approximate method of quantification based on their empirical observation of the quasi-parabolic shape of the absorbance peaks. The area enclosed by the derived absorbance parabola is linearly related to the product of the film contrast and the log of the molecular weight.
METHODS Construction and Calibration of the Analog Device An analog device was constructed to transform the transmittance output of a Beckman Model 25 spectrophotometer, equipped with a scanning accessory, by the appropriate hyperbolic function. The device incorporated a Model 433 J Programmable Multifunction Module (Analog Devices, P.O. Box 280, Norwood, MA 02062) with a simplified transfer function of eout = (10 mV/Ts)"^ where Tg represents the spectrophotometer output corresponding to transmission in millivolts. The exponent, ra, was adjusted by a ten-turn potentiometer to correspond to a contrast in the range of 0.2 to 5.0. The device interrupted the signal cable from the spectrophotometer to its 20 mV strip chart recorder, and acquired all signals and ± 15 V d.c. from the spectrophotometer external accessory connector. The device was ini tially calibrated in a test configuration and showed linear response of log (m) versus potentiometer setting for 0.51 < m < 1.67. The calibra tion was reverified upon installation on the spectrophotometer. A cir cuit diagram for this device may be obtained by writing to the authors.
BIOMEDICAL ENGINEERING II
383
Production of Fltiorograms Covalently closed circular supercoiled PM2 DNA was prepared (Legerski and o t h e r s , 1977) and digested to completion by a combination of Pst I and Hpa I restriction endonucleases as previously described (Legerski and others, 1978). The resulting fragments were electrophoresed at 3 V/cm in composite 2% Polyacrylamide - 0·5% agarose vertical slab gels (Peacock and Dingman, 1967) according to a reported procedure (Legerski and others, 1 9 7 8 ) . The gels were stained for 1 hr in a solution of 0.5 g/ml ethldium bromide and photographed during transillumination with 254 nm u.v. light. The u.v. source intensity, as Indicated by a Blak-Ray J-225 Short Wave u.v. Meter (Ultraviolet Products, Inc., San Gabriel, CA.) varied less than 10% along the long axis. Exposures of Kodak Royal Pan Film 4141 were made using a Graflex Graphex Camera through a u.v. cutoff filter and a No. 77A filter. Photographic and development conditions were adjusted to compensate for reciprocity failure according to the film manufacturer's specifications. The f-stop adjustment method (increased aperture with reduced developing time) was judged superior to the use of increased exposure time (compensated by decreased development time) and uncorrected (wide-aperture, shorter exposure time) photography conditions. Evaluation of Fluorograms Absorbance profiles were generated by scanning the negative with 546 nm light through a 0.05 mm slit. Absorbance peak heights and the masses of ab sorbance peaks excised from the chart paper provided independent determina tions of contrast when plotted versus log(molecular w e i g h t ) . Molecular weights were reverified by comparison with the mobilities of Hpa II re striction nuclease-generated fragments of pBR322 DNA, and varied little from reported values (Legerski and others, 1 9 7 8 ) . Contrasts were calculated from the linear least squares slope of the data. The peak height method showed the best linearity with log(molecular weight) and reproducibility. The analog device was adjusted to reflect the average contrast obtained from peak heights. The spectrophotomoter was adjusted to 100% transmittance through a transparent portion of the negative. Phototube dark current was nulled by blocking the slit and adjusting to 0% T. The device was switched in to provide the transformation and each of five lanes in a single slab gel was scanned. Strip chart tracings were excised and weighed. RESULTS AND DISCUSSION The results are summarized in Tables 1 and 2. Peak heights or areas are expressed as the percent of the total within each track. Errors in Table 1 represent the percentage error in the determination of each DNA band. TABLE 1 Precision of Relative Mass Measurements
Fragment
Actual %
Peak Height
Absorbance Peak Area
Transformed Peak Area
A Bl CI D El C2
31.3 24.1 16.4 13.9 7.6 6.7
25.1±2.9 % 23.0±3.2 % 18.6±4.3 % 16.9+4.3 % 9.2±11.9% 7.3+14.5%
24.6±2.1 % 21.8±4.5 % 19.0±5.0 % 17.6+6.0 % 9.2±19.4% 7.7+15.6%
32.1±4.1 % 24.5±4.3 % 17.1±7.5 % 14.8±7.3 % 6.4±17.4% 5.1±15.1%
Average Deviation
±6.85%
±9.27%
±9.28%
384
BIOMEDICAL ENGINEERING II TABLE 2 Accuracy of Relative Mass Measurements
Fragment
From Peak Height
A Bl CI D El C2 Average Error
Absorbance Peak Area
-19.8 % - 4.56% +13.4 % +21.6 % +21.1 % + 8.96%
-24.4 % - 9.54% +15.9 % +26.6 % +21.1 % +14.9 %
14.9%
18.2%
Transformed Peak Area -2.56% +1.66% +4.27% +6.47% -15.87% -23.9 % 9.11%
All the quantification methods evidenced acceptable precision. However, by the criterion of agreement with mobility-determined molecular weights, the analog transfer function is the most accurate method of mass determination examined with significant errors only for the lowest intensity bands. A signal attenuator would be a useful addition to the system since low contrast and high absorbance can drive the recorder off scale. Application of the theoretical principles discussed could be significantly expedited by direct digital manipulation of the absorbance scans. Construction of such a system is presently underway (Willis and Holmquist, unpublished). REFERENCES DeLeys, R.J., and D.A. Jackson (1976). Electrophoretlc analysis of covalently closed SV40 DNA: Boltzmann distribution of DNA species. Nucleic Acids Res., 2» 641-652. Depew R.E., and J.C. Wang (1975) Conformational fluctuations of DNA helix. Proc. Natl. Acad. Scl. U.S.A., 72^, 4275-4279. Dugalczyk, Α., H.W. Boyer and Η.Μ. Goodman (1975) Ligation of EcoRI endonuclease-generated DNA fragments into linear and circular struc tures. J. of Mol. Biol., 96, 171-184. Hörz, W., K.V. Oefele, and H. Schwab (1981) A simple logarithmic amplifier for the photographic quantitation of DNA in gel electrophoresis. Anal. Biochem., Π_, 266-270. Legerski, R.J., H.B. Gray, Jr., and D.L. Robberson (1977) A sensitive endonuclease probe for lesions in deoxyribonucleic acid helix structure produced by carcinogenic and mutagenic agents. J. Biol. Chem., 252, 8740-8746. Legerski, R.J., J.L. Hodnett, and H.B. Gray, Jr. (1978) Extracellular nucleases of Pseudomonas BAL 31. III. Use of the double-strand deoxyriboexonuclease activity as the basis of a convenient method for the mapping of fragments of DNA produced by cleavage with restriction enzjnnes. Nucleic Acids Res., _5> 1445-1464. Peacock, A.C. and C.W. Dingman (1967) Molecular weight estimation and separation of ribonucleic acids by electrophoresis in agarose acrylamide composite gels. Biochemistry 7, 668-674. Prunell, Α., F. Straus and B. Leblanc (1977) Photographic quantitation of DNA in gel electrophoresis. Anal. Biochem., 78, 57-65. Pulleyblank, D.E., M. Shure, D. Tang, J. Vinograd, and H.P. Vosberg (1975) Action of nicking-closing enzjrme on supercoiled and nonsupercolled closed circular DNA: formation of a Boltzmann distribution of topo logical Isomers. Proc. Natl. Acad. Scl. U.S.A., 72, 4280-4284. Pulleyblank, D.E., M. Shure, and J. Vinograd (1977) The quantitation of fluorescence by photography. Nucleic Acids Res., 4^, 1409-1418.
STAINED
Charles E. Willis*, Jaime A. Oro+ and Horace B. Gray, Jr.* *Department of Biochemical and Biophysical Sciences and +Department of Electrical Engineering University of Houston, Houston, Texas 77004
INTRODUCTION Fluorescence photographs constitute a permanent record of the results of electrophoretic separations of nucleic acid molecules. Images in these photographs are produced by enhanced ethldium bromide fluorescence when this dye is bound to DNA or RNA and exposed to u.v, light. Though fluorograms are used routinely to locate DNA In g e l s , nonlinearity in the pho tographic film darkening function complicates determination of the actual distribution of fluorescent material from photographic images. However, fluorescence photography is an inexpensive alternative to both direct fluorometry and radioactive labeling, and offers greater sensitivity for nucleic acid detection than direct absorbance measurements. Moreover, the principles of fluorographic analysis are applicable to autoradiography. The simplest methods for evaluating fluorographic images assume a linear relationship between the mass of fluorescing compound and the peak height of an absorbance scan of the photographic negative (DeLeys and Jackson, 1967). A similar method involves integration of the absorbance peaks (Depew and Wang, 1975; Dugaiczyk and others, 1 9 7 5 ) . Both approaches are valid as long as the maximum absorbance observed is much less than the contrast of the film (Pulleyblank and others, 1 9 7 5 ) . This is because there is a region of the film darkening curve, at relatively low incident intensities, in which absorbance of the developed film is linear with inci dent intensity. The range of useful measurement can be extended by deter mining the film darkening response as a function of incident intensity (Prunell and others, 1977; Pulleyblank and others, 1 9 7 7 ) . The absorbance traces are transformed into fluorescence intensity profiles (hence, mass distributions) by means of this film darkening curve in which the absor bance varies linearly with the log of exposure. Though the transformation can be performed by digital computers, one novel method (Hörz and others, 1981) describes the use of analog modification of densitometer output assuming the above linear-log film darkening function. Though inexpensive to implement, the analog method of Hörz and others (1981) uses the absorbance signal from the densitometer in the exponent of Its transfer function and as such is extremely sensitive to the determined
381
382
BIOMEDICAL ENGINEERING II
value of background absorbance. Expressing the transfer function in terms of transmittance makes use of the proportionality I « [ΐ/Τ)1/ϊ
(1)
where I^ is the intensity of film exposure, Ί_ represents fraction of inci dent light transmitted by the developed film and γ is the contrast of the film (slope of the linear region of the absorbance vs_ log(exposure) curve). This relationship is just as simple to implement and also has minimum and maximum calibration points for the transmittance signal. All conventional methods for determining the film darkening function rely on external fluorescent standards. Since an equimolar series of molecular weight standards is generally included in gel electrophoresis experiments with DNA, internal standardization is possible. In order to use these standards, assumptions must be made about the distribution of DNA within the band corresponding to a particular fragment. Assuming a Gaussian distribution of DNA within each band and effective diffusion coefficients which depend on the one-halfth power of molecular weight, the peak absor bance within each of these bands is linearly related to the product of the contrast of the film darkening curve and the log of the molecular weight of the DNA. The less that "diffusion" depends on molecular weight, the closer that the slope of a plot of peak absorbance versus log(molecular weight) approximates contrast. When the Gaussian distribution function and the linear-log darkening func tion are used to predict the absorbance as a function of position in the band, the absorbance profile describes a parabola. This relationship should be valid except at the "tails" where the linear-log function fails to describe the film darkening response. Pulleyblank and others (1977) have suggested an approximate method of quantification based on their empirical observation of the quasi-parabolic shape of the absorbance peaks. The area enclosed by the derived absorbance parabola is linearly related to the product of the film contrast and the log of the molecular weight.
METHODS Construction and Calibration of the Analog Device An analog device was constructed to transform the transmittance output of a Beckman Model 25 spectrophotometer, equipped with a scanning accessory, by the appropriate hyperbolic function. The device incorporated a Model 433 J Programmable Multifunction Module (Analog Devices, P.O. Box 280, Norwood, MA 02062) with a simplified transfer function of eout = (10 mV/Ts)"^ where Tg represents the spectrophotometer output corresponding to transmission in millivolts. The exponent, ra, was adjusted by a ten-turn potentiometer to correspond to a contrast in the range of 0.2 to 5.0. The device interrupted the signal cable from the spectrophotometer to its 20 mV strip chart recorder, and acquired all signals and ± 15 V d.c. from the spectrophotometer external accessory connector. The device was ini tially calibrated in a test configuration and showed linear response of log (m) versus potentiometer setting for 0.51 < m < 1.67. The calibra tion was reverified upon installation on the spectrophotometer. A cir cuit diagram for this device may be obtained by writing to the authors.
BIOMEDICAL ENGINEERING II
383
Production of Fltiorograms Covalently closed circular supercoiled PM2 DNA was prepared (Legerski and o t h e r s , 1977) and digested to completion by a combination of Pst I and Hpa I restriction endonucleases as previously described (Legerski and others, 1978). The resulting fragments were electrophoresed at 3 V/cm in composite 2% Polyacrylamide - 0·5% agarose vertical slab gels (Peacock and Dingman, 1967) according to a reported procedure (Legerski and others, 1 9 7 8 ) . The gels were stained for 1 hr in a solution of 0.5 g/ml ethldium bromide and photographed during transillumination with 254 nm u.v. light. The u.v. source intensity, as Indicated by a Blak-Ray J-225 Short Wave u.v. Meter (Ultraviolet Products, Inc., San Gabriel, CA.) varied less than 10% along the long axis. Exposures of Kodak Royal Pan Film 4141 were made using a Graflex Graphex Camera through a u.v. cutoff filter and a No. 77A filter. Photographic and development conditions were adjusted to compensate for reciprocity failure according to the film manufacturer's specifications. The f-stop adjustment method (increased aperture with reduced developing time) was judged superior to the use of increased exposure time (compensated by decreased development time) and uncorrected (wide-aperture, shorter exposure time) photography conditions. Evaluation of Fluorograms Absorbance profiles were generated by scanning the negative with 546 nm light through a 0.05 mm slit. Absorbance peak heights and the masses of ab sorbance peaks excised from the chart paper provided independent determina tions of contrast when plotted versus log(molecular w e i g h t ) . Molecular weights were reverified by comparison with the mobilities of Hpa II re striction nuclease-generated fragments of pBR322 DNA, and varied little from reported values (Legerski and others, 1 9 7 8 ) . Contrasts were calculated from the linear least squares slope of the data. The peak height method showed the best linearity with log(molecular weight) and reproducibility. The analog device was adjusted to reflect the average contrast obtained from peak heights. The spectrophotomoter was adjusted to 100% transmittance through a transparent portion of the negative. Phototube dark current was nulled by blocking the slit and adjusting to 0% T. The device was switched in to provide the transformation and each of five lanes in a single slab gel was scanned. Strip chart tracings were excised and weighed. RESULTS AND DISCUSSION The results are summarized in Tables 1 and 2. Peak heights or areas are expressed as the percent of the total within each track. Errors in Table 1 represent the percentage error in the determination of each DNA band. TABLE 1 Precision of Relative Mass Measurements
Fragment
Actual %
Peak Height
Absorbance Peak Area
Transformed Peak Area
A Bl CI D El C2
31.3 24.1 16.4 13.9 7.6 6.7
25.1±2.9 % 23.0±3.2 % 18.6±4.3 % 16.9+4.3 % 9.2±11.9% 7.3+14.5%
24.6±2.1 % 21.8±4.5 % 19.0±5.0 % 17.6+6.0 % 9.2±19.4% 7.7+15.6%
32.1±4.1 % 24.5±4.3 % 17.1±7.5 % 14.8±7.3 % 6.4±17.4% 5.1±15.1%
Average Deviation
±6.85%
±9.27%
±9.28%
384
BIOMEDICAL ENGINEERING II TABLE 2 Accuracy of Relative Mass Measurements
Fragment
From Peak Height
A Bl CI D El C2 Average Error
Absorbance Peak Area
-19.8 % - 4.56% +13.4 % +21.6 % +21.1 % + 8.96%
-24.4 % - 9.54% +15.9 % +26.6 % +21.1 % +14.9 %
14.9%
18.2%
Transformed Peak Area -2.56% +1.66% +4.27% +6.47% -15.87% -23.9 % 9.11%
All the quantification methods evidenced acceptable precision. However, by the criterion of agreement with mobility-determined molecular weights, the analog transfer function is the most accurate method of mass determination examined with significant errors only for the lowest intensity bands. A signal attenuator would be a useful addition to the system since low contrast and high absorbance can drive the recorder off scale. Application of the theoretical principles discussed could be significantly expedited by direct digital manipulation of the absorbance scans. Construction of such a system is presently underway (Willis and Holmquist, unpublished). REFERENCES DeLeys, R.J., and D.A. Jackson (1976). Electrophoretlc analysis of covalently closed SV40 DNA: Boltzmann distribution of DNA species. Nucleic Acids Res., 2» 641-652. Depew R.E., and J.C. Wang (1975) Conformational fluctuations of DNA helix. Proc. Natl. Acad. Scl. U.S.A., 72^, 4275-4279. Dugalczyk, Α., H.W. Boyer and Η.Μ. Goodman (1975) Ligation of EcoRI endonuclease-generated DNA fragments into linear and circular struc tures. J. of Mol. Biol., 96, 171-184. Hörz, W., K.V. Oefele, and H. Schwab (1981) A simple logarithmic amplifier for the photographic quantitation of DNA in gel electrophoresis. Anal. Biochem., Π_, 266-270. Legerski, R.J., H.B. Gray, Jr., and D.L. Robberson (1977) A sensitive endonuclease probe for lesions in deoxyribonucleic acid helix structure produced by carcinogenic and mutagenic agents. J. Biol. Chem., 252, 8740-8746. Legerski, R.J., J.L. Hodnett, and H.B. Gray, Jr. (1978) Extracellular nucleases of Pseudomonas BAL 31. III. Use of the double-strand deoxyriboexonuclease activity as the basis of a convenient method for the mapping of fragments of DNA produced by cleavage with restriction enzjnnes. Nucleic Acids Res., _5> 1445-1464. Peacock, A.C. and C.W. Dingman (1967) Molecular weight estimation and separation of ribonucleic acids by electrophoresis in agarose acrylamide composite gels. Biochemistry 7, 668-674. Prunell, Α., F. Straus and B. Leblanc (1977) Photographic quantitation of DNA in gel electrophoresis. Anal. Biochem., 78, 57-65. Pulleyblank, D.E., M. Shure, D. Tang, J. Vinograd, and H.P. Vosberg (1975) Action of nicking-closing enzjrme on supercoiled and nonsupercolled closed circular DNA: formation of a Boltzmann distribution of topo logical Isomers. Proc. Natl. Acad. Scl. U.S.A., 72, 4280-4284. Pulleyblank, D.E., M. Shure, and J. Vinograd (1977) The quantitation of fluorescence by photography. Nucleic Acids Res., 4^, 1409-1418.